SPADA Book - April 11, 2017

DRAFT ENVIRONMENTAL ORGANISMS PANEL, VERSION 5

2.2  73 74 In silico  screening shall be performed on all nucleic acid signature sequences used in the assay (e.g.,  75 primers, probes, amplicons, etc.) to demonstrate specificity to the target biological threat agent.  76 77 In silico  results are suggestive of potential performance issues, so will guide necessary additions to the  78 wet screening panels.   In silico  identification of potential cross‐reactions (false positives) or non‐ 79 verifications (false negatives) would require the affected organism/strain be included in the exclusivity  80 or inclusivity panels, respectively, if the strains are available.  81 82 A method developer‐selected tool to carry out the bioinformatics evaluation should be able to predict  83 hybridization events between signature components and a sequence in a database including available  84 genomic sequence data, databases and/or published documents describing the genetic sequences found  85 in soils that are representative of the regions of operation.  The selected tool should be able to identify  86 predicted hybridization events based on platform annealing temperatures, thus ensuring an accurate  87 degree of allowed mismatch is incorporated into predictions.  The program should detect possible  88 amplicons from any selected database of sequences.   89 90 Potential tools for in silico screening of nucleic acid sequences include:  91 92  http://sourceforge.net/projects/simulatepcr/files/?source=navbar  93 o This program will find all possible amplicons and real time fluorescing events from any  94 Bioinformatics Analyses 

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selected database of sequences. 

 NCBI tools 

The method developer submission should include:  

100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116

 Description of sequence databases used in the  in silico analysis 

 Description of conditions used for  in silico analysis 

o Stringency of  in silico analysis must match bench hybridization conditions

 Description of the tool(s) used for bioinformatics evaluation 

o Data demonstrating the selected tool(s) successfully predicts specificity that has been 

confirmed by wet‐lab testing on designated isolates  

 These data can be generated retrospectively using published assays 

 List of additional organisms and/or strains to be added to the inclusivity (Annex II) or exclusivity 

(Annex III) panels based on the bioinformatics evaluation